Recombinant DNA technology refers generally to the technique of integrating genetic information from a donor source into vectors for subsequent processing, such as through introduction into a host, whereby the transferred genetic information is copied and/or expressed in the new environment. Commonly, the genetic information exists in the form of complementary DNA (cDNA) derived from messenger RNA (mRNA) coding for a desired protein product. The carrier is frequently a plasmid having the capacity to incorporate cDNA for later replication in a host and, in some cases, actually to control expression of the cDNA and thereby direct synthesis of the encoded product in the host.
For some time, it has been known that the mammalian immune response is based on a series of complex cellular interactions, called the "immune network." Recent research has provided new insights into the inner workings of this network. While it remains clear that much of the response does, in fact, revolve around the network-like interactions of lymphocytes, macrophages, granulocytes, and other cells, immunologists now generally hold the opinion that soluble proteins, known as lymphokines, cytokines, or monokines, play a critical role in controlling these cellular interactions. Thus, there is considerable interest in the isolation, characterization, and mechanisms of action of cell modulatory factors, an understanding of which should lead to significant advancements in the diagnosis and therapy of numerous medical abnormalities, e.g., immune system disorders.
Lymphokines apparently mediate cellular activities in a variety of ways. They have been shown to support the proliferation, growth, and differentiation of the pluripotent hematopoietic stem cells into vast numbers of progenitors comprising diverse cellular lineages making up a complex immune system. These interactions between the cellular components are necessary for a healthy immune response. These different cellular lineages often respond in a different manner when lymphokines are administered in conjunction with other agents.
Cell lineages especially important to the immune response include two classes of lymphocytes: B-cells, which can produce and secrete immunoglobulins (proteins with the capability of recognizing and binding to foreign matter to effect its removal), and T-cells of various subsets that secrete lymphokines and induce or suppress the B-cells and various other cells (including other T-cells) making up the immune network.
Another important cell lineage is the mast cell (which has not been positively identified in all mammalian species), which is a granule-containing connective tissue cell located proximal to capillaries throughout the body. These cells are found in especially high concentrations in the lungs, skin, and gastrointestinal and genitourinary tracts. Mast cells play a central role in allergy-related disorders, particularly anaphylaxis as follows: when selected antigens crosslink one class of immunoglobulins bound to receptors on the mast cell surface, the mast cell degranulates and releases mediators, e.g., histamine, serotonin, heparin, and prostaglandins, which cause allergic reactions, e.g., anaphylaxis.
Research to better understand and thus potentially treat therapeutically various immune disorders has been hampered by the general inability to maintain cells of the immune system in vitro. Immunologists have discovered that culturing these cells can be accomplished through the use of T-cell and other cell supernatants, which contain various growth factors, including many of the lymphokines.
The detection, isolation, and purification of these factors are extremely difficult, being frequently complicated by the complexity of the supernatants they are typically located in, the divergencies and cross-overs of activities of the various components in the mixtures, the sensitivity (or lack thereof) of the assays utilized to ascertain the factors' properties, the frequent similarity in the range of molecular weights and other characteristics of the factors, and the very low concentration of the factors in their natural setting.
As more lymphokines become available, primarily through molecular cloning, interest has heightened in finding clinical applications for them. Because of physiological similarities to hormones (e.g., soluble factors, growth mediators, action via cell receptors), potential uses of lymphokines have been analogized to the current uses of hormones, e.g. Dexter, Nature, Vol. 321, pg. 198 (1986). One hope is that the levels of lymphokines in a patient can be manipulated directly or indirectly to bring about a beneficial immune response, e.g., suppression in the case of inflammation, allergy, or tissue rejection, or stimulation or potentiation in the case of infection or malignant growth. Other potential clinical uses of lymphokines include maintaining and expanding in vitro populations of certain immune system cells of one person for eventual reintroduction into the same or another person for a beneficial effect. For example, investigations are currently underway to determine whether populations of lymphokine-activated killer T cells of a patient can be expanded outside his or her body then reinjected to bring about an enhanced antitumor response. Another potential clinical use of lymphokines, particularly colony stimulating factors, such as granulocyte-macrophage colony stimulating factor (GM-CSF), and factors which enhance their activities, is in stimulating blood cell generation, for example, in pre-or post-chemotherapy or radiation therapy against tumors, in treatment of myeloid hypoplasias, or in treatment of neutrophil deficiency syndromes, Dexter, Nature Vol. 321, pg. 198 (1986). Another area where such factors would be useful is in bone marrow transplant therapy, which is being used increasingly to treat aplastic anemia and certain leukemias. Regulation of rejection mechanisms will be useful in organ transplantation situations.
There are two properties of lymphokines that have important consequences for such clinical applications: individual lymphokines are frequently pleiotropic, and the biological effects of one lymphokine can usually be modulated by at least one other lymphokine, either by inhibition or by potentiation. Thus, in many cases, addition of a lymphokine can affect diverse physiological responses, and combination therapies will often be advantageous over administration of a single lymphokine.
Most cytokines are not constitutively produced, but are produced after activation of the producer cells. Production after activation is typically only short-lived, usually for only a few days. Typically, cytokines are effective in the picogram to nanogram per ml range.
Table 4 from Feldman (1992) "Cytokines", pp. 438-440 in Roitt et al. (eds) The Encyclopedia of Immunology, Academic Press, New York, lists common cytokines and their predominant activities. More detailed descriptions are available from specific chapters in the Encyclopedia directed towards each individual cytokine.
TABLE 4 ______________________________________ common cytokines cytokine properties ______________________________________ IL-1.alpha. Activates lymphocytes, acute phase response, IL-1.beta. strongly proinflammatory, radioprotective IL-2 Activators of T, NK, B, macrophages IL-3 Multiple actions, hemopoietic cell growth; precursors of mast cells IL-4 B cell activating factor, T cell activator, growth and stimulating factor IL-5 B cell growth and differentiation factor, regulates production of granulocytes and macrophages IL-6 B cell differentiating factor, activation of T and B cells, induces acute phase response IL-7 Pre-B and pre-T cell growth factor, T cell growth factor IL-8 Involved in migration of neutrophils and T cells TNF-.alpha. Proinflammatory cytokine, induces acute phase response, thrombosis, cachexia TNF-.beta. as above (lympho- toxin) ______________________________________
A number of small inducible proteins secreted by leukocytes were reported by Brown et al. (1989) J. Immunol. 142:679-687. These proteins were described as part of a family of small, secreted, and inducible mouse and human proteins which are distantly related to a family of growth and inflammatory factors. It describes a class of "P600 induction-specific cDNA clones" but no biological activity was reported. No human equivalent had been isolated until now.
From the foregoing, it is evident that the discovery and development of new lymphokines could contribute to new therapies for a wide range of degenerative or abnormal conditions which directly or indirectly involve the immune system and/or hematopoietic cells. In particular, the discovery and development of lymphokines which enhance or potentiate the beneficial activities of known lymphokines would be highly advantageous. The present invention provides new interleukin compositions and related compounds, and methods for their use.